Multicolor Fluorescence In Situ Hybridization (FISH) Approaches for Simultaneous Analysis of the Entire Human Genome.

Analysis of the organization of the human genome is vital for understanding genetic diversity, human evolution, and disease pathogenesis. A number of approaches, such as multicolor fluorescence in situ hybridization (FISH) assays, cytogenomic microarray (CMA), and next-generation sequencing (NGS) technologies, are available for simultaneous analysis of the entire human genome. Multicolor FISH-based spectral karyotyping (SKY), multiplex FISH (M-FISH), and Rx-FISH may provide rapid identification of interchromosomal and intrachromosomal rearrangements as well as the origin of unidentified extrachromosomal elements. Recent advances in molecular cytogenetics have made it possible to efficiently examine the entire human genome in a single experiment at much higher resolution and specificity using CMA and NGS technologies. Here, we present an overview of the approaches available for genome-wide analyses. © 2018 by John Wiley & Sons, Inc.

Targeted deletion of a 170-kb cluster of LINE-1 repeats and implications for regional control.

Approximately half the mammalian genome is composed of repetitive sequences, and accumulating evidence suggests that some may have an impact on genome function. Here, we characterized a large array class of repeats of long-interspersed elements (LINE-1). Although widely distributed in mammals, locations of such arrays are species specific. Using targeted deletion, we asked whether a 170-kb LINE-1 array located at a mouse imprinted domain might function as a modulator of local transcriptional control. The LINE-1 array is lamina associated in differentiated ES cells consistent with its AT-richness, and although imprinting occurs both proximally and distally to the array, active LINE-1 transcripts within the tract are biallelically expressed. Upon deletion of the array, no perturbation of imprinting was observed, and abnormal phenotypes were not detected in maternal or paternal heterozygous or homozygous mutant mice. The array does not shield nonimprinted genes in the vicinity from local imprinting control. Reduced neural expression of protein-coding genes observed upon paternal transmission of the deletion is likely due to the removal of a brain-specific enhancer embedded within the LINE array. Our findings suggest that presence of a 170-kb LINE-1 array reflects the tolerance of the site for repeat insertion rather than an important genomic function in normal development.

Evolutionary dynamics of Anolis sex chromosomes revealed by sequencing of flow sorting-derived microchromosome-specific DNA.

Squamate reptiles show a striking diversity in modes of sex determination, including both genetic (XY or ZW) and temperature-dependent sex determination systems. The genomes of only a handful of species have been sequenced, analyzed and assembled including the genome of Anolis carolinensis. Despite a high genome coverage, only macrochromosomes of A. carolinensis were assembled whereas the content of most microchromosomes remained unclear. Most of the Anolis species have homomorphic XY sex chromosome system. However, some species have large heteromorphic XY chromosomes (e.g., A. sagrei) and even multiple sex chromosomes systems (e.g. A. pogus), that were shown to be derived from fusions of the ancestral XY with microautosomes. We applied next generation sequencing of flow sorting-derived chromosome-specific DNA pools to characterize the content and composition of microchromosomes in A. carolinensis and A. sagrei. Comparative analysis of sequenced chromosome-specific DNA pools revealed that the A. sagrei XY sex chromosomes contain regions homologous to several microautosomes of A. carolinensis. We suggest that the sex chromosomes of A. sagrei are derived by fusions of the ancestral sex chromosome with three microautosomes and subsequent loss of some genetic content on the Y chromosome.

Low rate of interchromosomal rearrangements during old radiation of gekkotan lizards (Squamata: Gekkota).

Gekkotan lizards are a highly specious (∼1600 described species) clade of squamate lizards with nearly cosmopolitan distribution in warmer areas. The clade is primarily nocturnal and forms an ecologically dominant part of the world nocturnal herpetofauna. However, molecular cytogenetic methods to study the evolution of karyotypes have not been widely applied in geckos. Our aim here was to uncover the extent of chromosomal rearrangements across the whole group Gekkota and to search for putative synapomorphies supporting the newly proposed phylogenetic relationships within this clade. We applied cross-species chromosome painting with the recently derived whole-chromosomal probes from the gekkonid species Gekko japonicus to members of the major gekkotan lineages. We included members of the families Diplodactylidae, Carphodactylidae, Pygopodidae, Eublepharidae, Phyllodactylidae and Gekkonidae. Our study demonstrates relatively high chromosome conservatism across the ancient group of gekkotan lizards. We documented that many changes in chromosomal shape across geckos can be attributed to intrachromosomal rearrangements. The documented rearrangements are not totally in agreement with the recently newly erected family Phyllodactylidae. The results also pointed to homoplasy, particularly in the reuse of chromosome breakpoints, in the evolution of gecko karyotypes.

Comparative analysis of sex chromosomes in Leporinus species (Teleostei, Characiformes) using chromosome painting.

BACKGROUND: The Leporinus genus, belonging to the Anostomidae family, is an interesting model for studies of sex chromosome evolution in fish, particularly because of the presence of heteromorphic sex chromosomes only in some species of the genus. In this study we used W chromosome-derived probes in a series of cross species chromosome painting experiments to try to understand events of sex chromosome evolution in this family. RESULTS: W chromosome painting probes from Leporinus elongatus, L. macrocephalus and L. obtusidens were hybridized to each others chromosomes. The results showed signals along their W chromosomes and the use of L. elongatus W probe against L. macrocephalus and L. obtusidens also showed signals over the Z chromosome. No signals were observed when the later aforementioned probe was used in hybridization procedures against other four Anostomidae species without sex chromosomes. CONCLUSIONS: Our results demonstrate a common origin of sex chromosomes in L. elongatus, L. macrocephalus and L. obtusidens but suggest that the L. elongatus chromosome system is at a different evolutionary stage. The absence of signals in the species without differentiated sex chromosomes does not exclude the possibility of cryptic sex chromosomes, but they must contain other Leporinus W sequences than those described here.

Uterine vasculature remodeling in human pregnancy involves functional macrochimerism by endothelial colony forming cells of fetal origin.

The potency of adult-derived circulating progenitor endothelial colony forming cells (ECFCs) is drastically surpassed by their fetal counterparts. Human pregnancy is associated with robust intensification of blood flow and vascular expansion in the uterus, crucial for placental perfusion and fetal supply. Here, we investigate whether fetal ECFCs transmigrate to maternal bloodstream and home to locations of maternal vasculogenesis, primarily the pregnant uterus. In the first instance, endothelial-like cells, originating from mouse fetuses expressing paternal eGFP, were identified within uterine endothelia. Subsequently, LacZ or enhanced green fluorescent protein (eGFP)-labeled human fetal ECFCs, transplanted into immunodeficient (NOD/SCID) fetuses on D15.5 pregnancy, showed similar integration into the mouse uterus by term. Mature endothelial controls (human umbilical vein endothelial cells), similarly introduced, were unequivocally absent. In humans, SRY was detected in 6 of 12 myometrial microvessels obtained from women delivering male babies. The copy number was calculated at 175 [IQR 149-471] fetal cells per millimeter square endothelium, constituting 12.5% of maternal vessel lumina. Cross-sections of similar human vessels, hybridized for Y-chromosome, positively identified endothelial-associated fetal cells. It appears that through ECFC donation, fetuses assist maternal uterine vascular expansion in pregnancy, potentiating placental perfusion and consequently their own fetal supply. In addition to fetal growth, this cellular mechanism holds implications for materno-fetal immune interactions and long-term maternal vascular health.

Conservation of chromosomes syntenic with avian autosomes in squamate reptiles revealed by comparative chromosome painting.

In contrast to mammals, birds exhibit a slow rate of chromosomal evolution. It is not clear whether high chromosome conservation is an evolutionary novelty of birds or was inherited from an earlier avian ancestor. The evolutionary conservatism of macrochromosomes between birds and turtles supports the latter possibility; however, the rate of chromosomal evolution is largely unknown in other sauropsids. In squamates, we previously reported strong conservatism of the chromosomes syntenic with the avian Z, which could reflect a peculiarity of this part of the genome. The chromosome 1 of iguanians and snakes is largely syntenic with chromosomes 3, 5 and 7 of the avian ancestral karyotype. In this project, we used comparative chromosome painting to determine how widely this synteny is conserved across nine families covering most of the main lineages of Squamata. The results suggest that the association of the avian ancestral chromosomes 3, 5 and 7 can be dated back to at least the early Jurassic and could be an ancestral characteristic for Unidentata (Serpentes, Iguania, Anguimorpha, Laterata and Scinciformata). In Squamata chromosome conservatism therefore also holds for the parts of the genome which are homologous to bird autosomes, and following on from this, a slow rate of chromosomal evolution could be a common characteristic of all sauropsids. The large evolutionary stasis in chromosome organization in birds therefore seems to be inherited from their ancestors, and it is particularly striking in comparison with mammals, probably the only major tetrapod lineage with an increased rate of chromosomal rearrangements as a whole.

Genomic restructuring in the Tasmanian devil facial tumour: chromosome painting and gene mapping provide clues to evolution of a transmissible tumour.

Devil facial tumour disease (DFTD) is a fatal, transmissible malignancy that threatens the world's largest marsupial carnivore, the Tasmanian devil, with extinction. First recognised in 1996, DFTD has had a catastrophic effect on wild devil numbers, and intense research efforts to understand and contain the disease have since demonstrated that the tumour is a clonal cell line transmitted by allograft. We used chromosome painting and gene mapping to deconstruct the DFTD karyotype and determine the chromosome and gene rearrangements involved in carcinogenesis. Chromosome painting on three different DFTD tumour strains determined the origins of marker chromosomes and provided a general overview of the rearrangement in DFTD karyotypes. Mapping of 105 BAC clones by fluorescence in situ hybridisation provided a finer level of resolution of genome rearrangements in DFTD strains. Our findings demonstrate that only limited regions of the genome, mainly chromosomes 1 and X, are rearranged in DFTD. Regions rearranged in DFTD are also highly rearranged between different marsupials. Differences between strains are limited, reflecting the unusually stable nature of DFTD. Finally, our detailed maps of both the devil and tumour karyotypes provide a physical framework for future genomic investigations into DFTD.

Chromosomal evolution in Gekkonidae. I. Chromosome painting between Gekko and Hemidactylus species reveals phylogenetic relationships within the group.

Geckos are a large group of lizards characterized by a rich variety of species, different modes of sex determination and diverse karyotypes. In spite of many unresolved questions on lizards' phylogeny and taxonomy, the karyotypes of most geckos have been studied by conventional cytogenetic methods only. We used flow-sorted chromosome-specific painting probes of Japanese gecko (Gekko japonicus), Mediterranean house gecko (Hemidactylus turcicus) and flat-tailed house gecko (Hemidactylus platyurus) to reveal homologous regions and to study karyotype evolution in seven gecko species (Gekko gecko, G. japonicus, G. ulikovskii, G. vittatus, Hemidactylus frenatus, H. platyurus and H. turcicus). Generally, the karyotypes of geckos were found to be conserved, but we revealed some characteristic rearrangements including both fissions and fusions in Hemidactylus. The karyotype of H. platyurus contained a heteromorphic pair in all female individuals, where one of the homologues had a terminal DAPI-negative and C-positive heterochromatic block that might indicate a putative sex chromosome. Among two male individuals studied, only one carried such a polymorphism, and the second one had none, suggesting a possible ZZ/ZW sex determination in some populations of this species. We found that all Gekko species have retained the putative ancestral karyotype, whilst the fission of the largest ancestral chromosome occurred in the ancestor of modern Hemidactylus species. Three common fissions occurred in the ancestor of Mediterranean house and flat-tailed house geckos, suggesting their sister group relationships. PCR-assisted mapping on flow-sorted chromosome libraries with conserved DMRT1 gene primers in G. japonicus indicates the localization of DMRT1 gene on chromosome 6.

A first-generation integrated tammar wallaby map and its use in creating a tammar wallaby first-generation virtual genome map.

BACKGROUND: The limited (2X) coverage of the tammar wallaby (Macropus eugenii) genome sequence dataset currently presents a challenge for assembly and anchoring onto chromosomes. To provide a framework for this assembly, it would be a great advantage to have a dense map of the tammar wallaby genome. However, only limited mapping data are available for this non-model species, comprising a physical map and a linkage map. RESULTS: We combined all available tammar wallaby mapping data to create a tammar wallaby integrated map, using the Location DataBase (LDB) strategy. This first-generation integrated map combines all available information from the second-generation tammar wallaby linkage map with 148 loci, and extensive FISH mapping data for 492 loci, especially for genes likely to be located at the ends of wallaby chromosomes or at evolutionary breakpoints inferred from comparative information. For loci whose positions are only approximately known, their location in the integrated map was refined on the basis of comparative information from opossum (Monodelphis domestica) and human. Interpolation of segments from the opossum and human assemblies into the integrated map enabled the subsequent construction of a tammar wallaby first-generation virtual genome map, which comprises 14336 markers, including 13783 genes recruited from opossum and human assemblies. Both maps are freely available at http://compldb.angis.org.au. CONCLUSIONS: The first-generation integrated map and the first-generation virtual genome map provide a backbone for the chromosome assembly of the tammar wallaby genome sequence. For example, 78% of the 10257 gene-scaffolds in the Ensembl annotation of the tammar wallaby genome sequence (including 10522 protein-coding genes) can now be given a chromosome location in the tammar wallaby virtual genome map.

Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.

BACKGROUND: We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. RESULTS: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. CONCLUSIONS: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.

Strong conservation of the bird Z chromosome in reptilian genomes is revealed by comparative painting despite 275 million years divergence.

The divergence of lineages leading to extant squamate reptiles (lizards, snakes, and amphisbaenians) and birds occurred about 275 million years ago. Birds, unlike squamates, have karyotypes that are typified by the presence of a number of very small chromosomes. Hence, a number of chromosome rearrangements might be expected between bird and squamate genomes. We used chromosome-specific DNA from flow-sorted chicken (Gallus gallus) Z sex chromosomes as a probe in cross-species hybridization to metaphase spreads of 28 species from 17 families representing most main squamate lineages and single species of crocodiles and turtles. In all but one case, the Z chromosome was conserved intact despite very ancient divergence of sauropsid lineages. Furthermore, the probe painted an autosomal region in seven species from our sample with characterized sex chromosomes, and this provides evidence against an ancestral avian-like system of sex determination in Squamata. The avian Z chromosome synteny is, therefore, conserved albeit it is not a sex chromosome in these squamate species.

Differentiation of sex chromosomes and karyotypic evolution in the eye-lid geckos (Squamata: Gekkota: Eublepharidae), a group with different modes of sex determination.

The eyelid geckos (family Eublepharidae) include both species with temperature-dependent sex determination and species where genotypic sex determination (GSD) was suggested based on the observation of equal sex ratios at several incubation temperatures. In this study, we present data on karyotypes and chromosomal characteristics in 12 species (Aeluroscalabotes felinus, Coleonyx brevis, Coleonyx elegans, Coleonyx variegatus, Eublepharis angramainyu, Eublepharis macularius, Goniurosaurus araneus, Goniurosaurus lichtenfelderi, Goniurosaurus luii, Goniurosaurus splendens, Hemitheconyx caudicinctus, and Holodactylus africanus) covering all genera of the family, and search for the presence of heteromorphic sex chromosomes. Phylogenetic mapping of chromosomal changes showed a long evolutionary stasis of karyotypes with all acrocentric chromosomes followed by numerous chromosomal rearrangements in the ancestors of two lineages. We have found heteromorphic sex chromosomes in only one species, which suggests that sex chromosomes in most GSD species of the eyelid geckos are not morphologically differentiated. The sexual difference in karyotype was detected only in C. elegans which has a multiple sex chromosome system (X(1)X(2)Y). The metacentric Y chromosome evolved most likely via centric fusion of two acrocentric chromosomes involving loss of interstitial telomeric sequences. We conclude that the eyelid geckos exhibit diversity in sex determination ranging from the absence of any sexual differences to heteromorphic sex chromosomes, which makes them an interesting system for exploring the evolutionary origin of sexually dimorphic genomes.

Epigenetic modifications on X chromosomes in marsupial and monotreme mammals and implications for evolution of dosage compensation.

X chromosome dosage compensation in female eutherian mammals is regulated by the noncoding Xist RNA and is associated with the differential acquisition of active and repressive histone modifications, resulting in repression of most genes on one of the two X chromosome homologs. Marsupial mammals exhibit dosage compensation; however, they lack Xist, and the mechanisms conferring epigenetic control of X chromosome dosage compensation remain elusive. Oviparous mammals, the monotremes, have multiple X chromosomes, and it is not clear whether they undergo dosage compensation and whether there is epigenetic dimorphism between homologous pairs in female monotremes. Here, using antibodies against DNA methylation, eight different histone modifications, and HP1, we conduct immunofluorescence on somatic cells of the female Australian marsupial possum Trichosurus vulpecula, the female platypus Ornithorhynchus anatinus, and control mouse cells. The two marsupial X's were different for all epigenetic features tested. In particular, unlike in the mouse, both repressive modifications, H3K9me3 and H4K20Me3, are enriched on one of the X chromosomes, and this is associated with the presence of HP1 and hypomethylation of DNA. Using sequential labeling, we determine that this DNA hypomethylated X correlates with histone marks of inactivity. These results suggest that female marsupials use a repressive histone-mediated inactivation mechanism and that this may represent an ancestral dosage compensation process that differs from eutherians that require Xist transcription and DNA methylation. In comparison to the marsupial, the monotreme exhibited no epigenetic differences between homologous X chromosomes, suggesting the absence of a dosage compensation process comparable to that in therians.

Aneuploidy in mitosis of PtK1 cells is generated by random loss and nondisjunction of individual chromosomes.

Chromosome lagging at anaphase and migration of both sister chromatids to the same pole, i.e. nondisjunction, are two chromosome-segregation errors producing aneuploid cell progeny. Here, we developed an assay for the simultaneous detection of both chromosome-segregation errors in the marsupial PtK1 cell line by using multiplex fluorescence in situ hybridization with specific painting probes obtained by chromosome flow sorting. No differential susceptibility of the six PtK1 chromosomes to undergo nondisjunction and/or chromosome loss was observed in ana-telophase cells recovering from a nocodazole- or a monastrol-induced mitotic arrest, suggesting that the recurrent presence of specific chromosomes in several cancer types reflects selection effects rather than differential propensities of specific chromosomes to undergo missegregation. Experiments prolonging metaphase duration during drug recovery and inhibiting Aurora-B kinase activity on metaphase-aligned chromosomes provided evidence that some type of merotelic orientations was involved in the origin of both chromosome-segregation errors. Visualization of mero-syntelic kinetochore-microtubule attachments (a merotelic kinetochore in which the thicker microtubule bundle is attached to the same pole to which the sister kinetochore is connected) identified a peculiar malorientation that might participate in the generation of nondisjunction. Our findings imply random missegregation of chromosomes as the initial event in the generation of aneuploidy in mammalian somatic cells.

Perturbation of T-cell development by insertional mutation of a PrP transgene.

The normal cellular form of the prion protein PrP(C) is a glycosylphosphatidylinositol-linked cell-surface glycoprotein expressed primarily by cells of the nervous and immune systems. There is evidence to suggest that PrP(C) is involved in cell signalling and cellular homeostasis. We have investigated the immune composition of peripheral lymphoid tissue in PrP-/-, wild-type, tg19 and tga20 strains of mice, which express 0, 1-, 3-5- and 4-7-fold higher levels of PrP(C), respectively, relative to wild-type mice. Our data show that tga20 mice have a reduced number of spleen T-cell receptor (TCR)-alphabeta(+) T cells and an increased number of TCR-gammadelta(+) T cells compared with wild-type mice. This was not seen in tg19 mice, which also express elevated levels of PrP(C). In addition, we have found that the Prnp transgene in the tga20 genome is located centrally on chromosome 17, in or around genes involved in T-cell development. Significantly, mRNA transcripts from pre-TCR-alpha (pTalpha), a T-cell development gene located on mouse chromosome 17, are drastically reduced in tga20 mice, indicative of a perturbation in pTalpha gene regulation. We propose that the immune cell phenotype of tga20 mice may be caused by the insertional mutation of the Prnp transgene into the pTalpha gene or its regulatory elements.

Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes.

In therian mammals (placentals and marsupials), sex is determined by an XX female: XY male system, in which a gene (SRY) on the Y affects male determination. There is no equivalent in other amniotes, although some taxa (notably birds and snakes) have differentiated sex chromosomes. Birds have a ZW female: ZZ male system with no homology with mammal sex chromosomes, in which dosage of a Z-borne gene (possibly DMRT1) affects male determination. As the most basal mammal group, the egg-laying monotremes are ideal for determining how the therian XY system evolved. The platypus has an extraordinary sex chromosome complex, in which five X and five Y chromosomes pair in a translocation chain of alternating X and Y chromosomes. We used physical mapping to identify genes on the pairing regions between adjacent X and Y chromosomes. Most significantly, comparative mapping shows that, contrary to earlier reports, there is no homology between the platypus and therian X chromosomes. Orthologs of genes in the conserved region of the human X (including SOX3, the gene from which SRY evolved) all map to platypus chromosome 6, which therefore represents the ancestral autosome from which the therian X and Y pair derived. Rather, the platypus X chromosomes have substantial homology with the bird Z chromosome (including DMRT1) and to segments syntenic with this region in the human genome. Thus, platypus sex chromosomes have strong homology with bird, but not to therian sex chromosomes, implying that the therian X and Y chromosomes (and the SRY gene) evolved from an autosomal pair after the divergence of monotremes only 166 million years ago. Therefore, the therian X and Y are more than 145 million years younger than previously thought.

Chromhome: a rich internet application for accessing comparative chromosome homology maps.

BACKGROUND: Comparative genomics has become a significant research area in recent years, following the availability of a number of sequenced genomes. The comparison of genomes is of great importance in the analysis of functionally important genome regions. It can also be used to understand the phylogenetic relationships of species and the mechanisms leading to rearrangement of karyotypes during evolution. Many species have been studied at the cytogenetic level by cross species chromosome painting. With the large amount of such information, it has become vital to computerize the data and make them accessible worldwide. Chromhome http://www.chromhome.org is a comprehensive web application that is designed to provide cytogenetic comparisons among species and to fulfil this need. RESULTS: The Chromhome application architecture is multi-tiered with an interactive client layer, business logic and database layers. Enterprise java platform with open source framework OpenLaszlo is used to implement the Rich Internet Chromhome Application. Cross species comparative mapping raw data are collected and the processed information is stored into MySQL Chromhome database. Chromhome Release 1.0 contains 109 homology maps from 51 species. The data cover species from 14 orders and 30 families. The homology map displays all the chromosomes of the compared species as one image, making comparisons among species easier. Inferred data also provides maps of homologous regions that could serve as a guideline for researchers involved in phylogenetic or evolution based studies. CONCLUSION: Chromhome provides a useful resource for comparative genomics, holding graphical homology maps of a wide range of species. It brings together cytogenetic data of many genomes under one roof. Inferred painting can often determine the chromosomal homologous regions between two species, if each has been compared with a common third species. Inferred painting greatly reduces the need to map entire genomes and helps focus only on relevant regions of the chromosomes of the species under study. Future releases of Chromhome will accommodate more species and their respective gene and BAC maps, in addition to chromosome painting data. Chromhome application provides a single-page interface (SPI) with desktop style layout, delivering a better and richer user experience.

Genomic instability within centromeres of interspecific marsupial hybrids.

Several lines of evidence suggest that, within a lineage, particular genomic regions are subject to instability that can lead to specific types of chromosome rearrangements important in species incompatibility. Within family Macropodidae (kangaroos, wallabies, bettongs, and potoroos), which exhibit recent and extensive karyotypic evolution, rearrangements involve chiefly the centromere. We propose that centromeres are the primary target for destabilization in cases of genomic instability, such as interspecific hybridization, and participate in the formation of novel chromosome rearrangements. Here we use standard cytological staining, cross-species chromosome painting, DNA probe analyses, and scanning electron microscopy to examine four interspecific macropodid hybrids (Macropus rufogriseus x Macropus agilis). The parental complements share the same centric fusions relative to the presumed macropodid ancestral karyotype, but can be differentiated on the basis of heterochromatic content, M. rufogriseus having larger centromeres with large C-banding positive regions. All hybrids exhibited the same pattern of chromosomal instability and remodeling specifically within the centromeres derived from the maternal (M. rufogriseus) complement. This instability included amplification of a satellite repeat and a transposable element, changes in chromatin structure, and de novo whole-arm rearrangements. We discuss possible reasons and mechanisms for the centromeric instability and remodeling observed in all four macropodid hybrids.

The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z.

BACKGROUND: Sex-determining systems have evolved independently in vertebrates. Placental mammals and marsupials have an XY system, birds have a ZW system. Reptiles and amphibians have different systems, including temperature-dependent sex determination, and XY and ZW systems that differ in origin from birds and placental mammals. Monotremes diverged early in mammalian evolution, just after the mammalian clade diverged from the sauropsid clade. Our previous studies showed that male platypus has five X and five Y chromosomes, no SRY, and DMRT1 on an X chromosome. In order to investigate monotreme sex chromosome evolution, we performed a comparative study of platypus and echidna by chromosome painting and comparative gene mapping. RESULTS: Chromosome painting reveals a meiotic chain of nine sex chromosomes in the male echidna and establishes their order in the chain. Two of those differ from those in the platypus, three of the platypus sex chromosomes differ from those of the echidna and the order of several chromosomes is rearranged. Comparative gene mapping shows that, in addition to bird autosome regions, regions of bird Z chromosomes are homologous to regions in four platypus X chromosomes, that is, X1, X2, X3, X5, and in chromosome Y1. CONCLUSION: Monotreme sex chromosomes are easiest to explain on the hypothesis that autosomes were added sequentially to the translocation chain, with the final additions after platypus and echidna divergence. Genome sequencing and contig anchoring show no homology yet between platypus and therian Xs; thus, monotremes have a unique XY sex chromosome system that shares some homology with the avian Z.